Decitabine cytotoxicity is promoted by dCMP deaminase DCTD and mitigated by SUMO-dependent E3 ligase TOPORS.

The nucleoside analogue decitabine (or 5-aza-dC) is used to treat several haematological cancers. Upon its triphosphorylation and incorporation into DNA, 5-aza-dC induces covalent DNA methyltransferase 1 DNA-protein crosslinks (DNMT1-DPCs), leading to DNA hypomethylation. However, 5-aza-dC's clinical outcomes vary, and relapse is common. Using genome-scale CRISPR/Cas9 screens, we map factors determining 5-aza-dC sensitivity. Unexpectedly, we find that loss of the dCMP deaminase DCTD causes 5-aza-dC resistance, suggesting that 5-aza-dUMP generation is cytotoxic. Combining results from a subsequent genetic screen in DCTD-deficient cells with the identification of the DNMT1-DPC-proximal proteome, we uncover the ubiquitin and SUMO1 E3 ligase, TOPORS, as a new DPC repair factor. TOPORS is recruited to SUMOylated DNMT1-DPCs and promotes their degradation. Our study suggests that 5-aza-dC-induced DPCs cause cytotoxicity when DPC repair is compromised, while cytotoxicity in wild-type cells arises from perturbed nucleotide metabolism, potentially laying the foundations for future identification of predictive biomarkers for decitabine treatment.

RAD54L2 counters TOP2-DNA adducts to promote genome stability.

The catalytic cycle of topoisomerase 2 (TOP2) enzymes proceeds via a transient DNA double-strand break (DSB) intermediate termed the TOP2 cleavage complex (TOP2cc), in which the TOP2 protein is covalently bound to DNA. Anticancer agents such as etoposide operate by stabilizing TOP2ccs, ultimately generating genotoxic TOP2-DNA protein cross-links that require processing and repair. Here, we identify RAD54 like 2 (RAD54L2) as a factor promoting TOP2cc resolution. We demonstrate that RAD54L2 acts through a novel mechanism together with zinc finger protein associated with tyrosyl-DNA phosphodiesterase 2 (TDP2) and TOP2 (ZATT/ZNF451) and independent of TDP2. Our work suggests a model wherein RAD54L2 recognizes sumoylated TOP2 and, using its ATPase activity, promotes TOP2cc resolution and prevents DSB exposure. These findings suggest RAD54L2-mediated TOP2cc resolution as a potential mechanism for cancer therapy resistance and highlight RAD54L2 as an attractive candidate for drug discovery.

Large-scale exome sequence analysis identifies sex- and age-specific determinants of obesity.

Obesity contributes substantially to the global burden of disease and has a significant heritable component. Recent large-scale exome sequencing studies identified several genes in which rare, protein-coding variants have large effects on adult body mass index (BMI). Here we extended such work by performing sex-stratified associations in the UK Biobank study (N∼420,000). We identified genes in which rare heterozygous loss-of-function increases adult BMI in women (DIDO1, PTPRG, and SLC12A5) and in men (SLTM), with effect sizes up to ∼8 kg/m2. This is complemented by analyses implicating rare variants in OBSCN and MADD for recalled childhood adiposity. The known functions of these genes, as well as findings of common variant genome-wide pathway enrichment analyses, suggest a role for neuron death, apoptosis, and DNA damage response mechanisms in the susceptibility to obesity across the life-course. These findings highlight the importance of considering sex-specific and life-course effects in the genetic regulation of obesity.

Inactivating TDP2 missense mutation in siblings with congenital abnormalities reminiscent of fanconi anemia.

Mutations in TDP2, encoding tyrosyl-DNA phosphodiesterase 2, have been associated with a syndromal form of autosomal recessive spinocerebellar ataxia, type 23 (SCAR23). This is a very rare and progressive neurodegenerative disorder described in only nine patients to date, and caused by splice site or nonsense mutations that result in greatly reduced or absent TDP2 protein. TDP2 is required for the rapid repair of DNA double-strand breaks induced by abortive DNA topoisomerase II (TOP2) activity, important for genetic stability in post-mitotic cells such as neurons. Here, we describe a sibship that is homozygous for the first TDP2 missense mutation (p.Glu152Lys) and which presents with clinical features overlapping both SCAR23 and Fanconi anemia (FA). We show that in contrast to previously reported SCAR23 patients, fibroblasts derived from the current patient retain significant levels of TDP2 protein. However, this protein is catalytically inactive, resulting in reduced rates of repair of TOP2-induced DNA double-strand breaks and cellular hypersensitivity to the TOP2 poison, etoposide. The TDP2-mutated patient-derived fibroblasts do not display increased chromosome breakage following treatment with DNA crosslinking agents, but both TDP2-mutated and FA cells exhibit increased chromosome breakage in response to etoposide. This suggests that the FA pathway is required in response to TOP2-induced DNA lesions, providing a possible explanation for the clinical overlap between FA and the current TDP2-mutated patients. When reviewing the relatively small number of patients with SCAR23 that have been reported, it is clear that the phenotype of such patients can extend beyond neurological features, indicating that the TDP2 protein influences not only neural homeostasis but also other tissues as well.

Combinatorial CRISPR screen identifies fitness effects of gene paralogues.

Genetic redundancy has evolved as a way for human cells to survive the loss of genes that are single copy and essential in other organisms, but also allows tumours to survive despite having highly rearranged genomes. In this study we CRISPR screen 1191 gene pairs, including paralogues and known and predicted synthetic lethal interactions to identify 105 gene combinations whose co-disruption results in a loss of cellular fitness. 27 pairs influence fitness across multiple cell lines including the paralogues FAM50A/FAM50B, two genes of unknown function. Silencing of FAM50B occurs across a range of tumour types and in this context disruption of FAM50A reduces cellular fitness whilst promoting micronucleus formation and extensive perturbation of transcriptional programmes. Our studies reveal the fitness effects of FAM50A/FAM50B in cancer cells.

Characterization of a novel loss-of-function variant in TDP2 in two adult patients with spinocerebellar ataxia autosomal recessive 23 (SCAR23).

TDP2 encodes a 5'-tyrosyl DNA phosphodiesterase required for the efficient repair of double-strand breaks (DSBs) induced by the abortive activity of DNA topoisomerase II (TOP2). To date, only three homozygous variants in TDP2 have been reported in six patients from four unrelated pedigrees with spinocerebellar ataxia 23 (SCAR23). By whole-exome sequencing, we identified a novel TDP2 splice-site variant (c.636 + 3_636 + 6del) in two Italian siblings (aged 40 and 45) showing progressive ataxia, intellectual disability, speech delay, refractory seizures, and various physical anomalies. The variant caused exon 5 skipping with consequent nonsense-mediated mRNA decay and defective repair of TOP2-induced DSBs, as demonstrated by the functional assays on the patients' fibroblasts. Our findings further demonstrate the pathogenic role of TDP2 biallelic loss-of-function variants in SCAR23 pathogenesis. Considering the age of our patients, the oldest reported to date, and their extensive follow-up, our study delineates in more detail the clinical phenotype related to the loss of TDP2 activity.

Untangling trapped topoisomerases with tyrosyl-DNA phosphodiesterases.

DNA topoisomerases alleviate the torsional stress that is generated by processes that are central to genome metabolism such as transcription and DNA replication. To do so, these enzymes generate an enzyme intermediate known as the cleavage complex in which the topoisomerase is covalently linked to the termini of a DNA single- or double-strand break. Whilst cleavage complexes are normally transient they can occasionally become abortive, creating protein-linked DNA breaks that threaten genome stability and cell survival; a process promoted and exploited in the cancer clinic by the use of topoisomerase 'poisons'. Here, we review the consequences to genome stability and human health of abortive topoisomerase-induced DNA breakage and the cellular pathways that cells have adopted to mitigate them, with particular focus on an important class of enzymes known as tyrosyl-DNA phosphodiesterases.

Effects of TDP2/VPg Unlinkase Activity on Picornavirus Infections Downstream of Virus Translation.

In this study, we characterized the role of host cell protein tyrosyl-DNA phosphodiesterase 2 (TDP2) activity, also known as VPg unlinkase, in picornavirus infections in a human cell model of infection. TDP2/VPg unlinkase is used by picornaviruses to remove the small polypeptide, VPg (Virus Protein genome-linked, the primer for viral RNA synthesis), from virus genomic RNA. We utilized a CRISPR/Cas-9-generated TDP2 knock out (KO) human retinal pigment epithelial-1 (hRPE-1) cell line, in addition to the wild type (WT) counterpart for our studies. We determined that in the absence of TDP2, virus growth kinetics for two enteroviruses (poliovirus and coxsackievirus B3) were delayed by about 2 h. Virus titers were reduced by ~2 log10 units for poliovirus and 0.5 log10 units for coxsackievirus at 4 hours post-infection (hpi), and by ~1 log10 unit at 6 hpi for poliovirus. However, virus titers were nearly indistinguishable from those of control cells by the end of the infectious cycle. We determined that this was not the result of an alternative source of VPg unlinkase activity being activated in the absence of TPD2 at late times of infection. Viral protein production in TDP2 KO cells was also substantially reduced at 4 hpi for poliovirus infection, consistent with the observed growth kinetics delay, but reached normal levels by 6 hpi. Interestingly, this result differs somewhat from what has been reported previously for the TDP2 KO mouse cell model, suggesting that either cell type or species-specific differences might be playing a role in the observed phenotype. We also determined that catalytically inactive TDP2 does not rescue the growth defect, confirming that TDP2 5' phosphodiesterase activity is required for efficient virus replication. Importantly, we show for the first time that polysomes can assemble efficiently on VPg-linked RNA after the initial round of translation in a cell culture model, but both positive and negative strand RNA production is impaired in the absence of TDP2 at mid-times of infection, indicating that the presence of VPg on the viral RNA affects a step in the replication cycle downstream of translation (e.g., RNA synthesis). In agreement with this conclusion, we found that double-stranded RNA production (a marker of viral RNA synthesis) is delayed in TDP2 KO RPE-1 cells. Moreover, we show that premature encapsidation of nascent, VPg-linked RNA is not responsible for the observed virus growth defect. Our studies provide the first lines of evidence to suggest that either negative- or positive-strand RNA synthesis (or both) is a likely candidate for the step that requires the removal of VPg from the RNA for an enterovirus infection to proceed efficiently.

Topoisomerase II-Induced Chromosome Breakage and Translocation Is Determined by Chromosome Architecture and Transcriptional Activity.

Topoisomerase II (TOP2) relieves torsional stress by forming transient cleavage complex intermediates (TOP2ccs) that contain TOP2-linked DNA breaks (DSBs). While TOP2ccs are normally reversible, they can be "trapped" by chemotherapeutic drugs such as etoposide and subsequently converted into irreversible TOP2-linked DSBs. Here, we have quantified etoposide-induced trapping of TOP2ccs, their conversion into irreversible TOP2-linked DSBs, and their processing during DNA repair genome-wide, as a function of time. We find that while TOP2 chromatin localization and trapping is independent of transcription, it requires pre-existing binding of cohesin to DNA. In contrast, the conversion of trapped TOP2ccs to irreversible DSBs during DNA repair is accelerated 2-fold at transcribed loci relative to non-transcribed loci. This conversion is dependent on proteasomal degradation and TDP2 phosphodiesterase activity. Quantitative modeling shows that only two features of pre-existing chromatin structure-namely, cohesin binding and transcriptional activity-can be used to predict the kinetics of TOP2-induced DSBs.

Confirming TDP2 mutation in spinocerebellar ataxia autosomal recessive 23 (SCAR23).

Objective: To address the relationship between mutations in the DNA strand break repair protein tyrosyl DNA phosphodiesterase 2 (TDP2) and spinocerebellar ataxia autosomal recessive 23 (SCAR23) and to characterize the cellular phenotype of primary fibroblasts from this disease. Methods: We have used exome sequencing, Sanger sequencing, gene editing and cell biology, biochemistry, and subcellular mitochondrial analyses for this study. Results: We have identified a patient in the United States with SCAR23 harboring the same homozygous TDP2 mutation as previously reported in 3 Irish siblings (c.425+1G>A). The current and Irish patients share the same disease haplotype, but the current patient lacks a homozygous variant present in the Irish siblings in the closely linked gene ZNF193, eliminating this as a contributor to the disease. The current patient also displays symptoms consistent with mitochondrial dysfunction, although levels of mitochondrial function in patient primary skin fibroblasts are normal. However, we demonstrate an inability in patient primary fibroblasts to rapidly repair topoisomerase-induced DNA double-strand breaks (DSBs) in the nucleus and profound hypersensitivity to this type of DNA damage. Conclusions: These data confirm the TDP2 mutation as causative for SCAR23 and highlight the link between defects in nuclear DNA DSB repair, developmental delay, epilepsy, and ataxia.

TDP2, TOP2, and SUMO: what is ZATT about?

Recently, ZATT (also known as ZNF451 or Zpf451) was reported by Schellenberg et al. to aid the removal of Topoisomerase II cleavage complexes by stimulating the phosphodiesterase activity of Tyrosyl DNA Phosphodiesterase 2. Although the full implication of this discovery is unknown, it will help us understand how cells respond to topoisomerase-induced genome damage and chemotherapeutic topoisomerase 'poisons'.

TDP2 suppresses chromosomal translocations induced by DNA topoisomerase II during gene transcription.

DNA double-strand breaks (DSBs) induced by abortive topoisomerase II (TOP2) activity are a potential source of genome instability and chromosome translocation. TOP2-induced DNA double-strand breaks are rejoined in part by tyrosyl-DNA phosphodiesterase 2 (TDP2)-dependent non-homologous end-joining (NHEJ), but whether this process suppresses or promotes TOP2-induced translocations is unclear. Here, we show that TDP2 rejoins DSBs induced during transcription-dependent TOP2 activity in breast cancer cells and at the translocation 'hotspot', MLL. Moreover, we find that TDP2 suppresses chromosome rearrangements induced by TOP2 and reduces TOP2-induced chromosome translocations that arise during gene transcription. Interestingly, however, we implicate TDP2-dependent NHEJ in the formation of a rare subclass of translocations associated previously with therapy-related leukemia and characterized by junction sequences with 4-bp of perfect homology. Collectively, these data highlight the threat posed by TOP2-induced DSBs during transcription and demonstrate the importance of TDP2-dependent non-homologous end-joining in protecting both gene transcription and genome stability.DNA double-strand breaks (DSBs) induced by topoisomerase II (TOP2) are rejoined by TDP2-dependent non-homologous end-joining (NHEJ) but whether this promotes or suppresses translocations is not clear. Here the authors show that TDP2 suppresses chromosome translocations from DSBs introduced during gene transcription.